Alpha-farnesene (FIG. 1) is an acyclic sesquiterpene hydrocarbon (C15H24; 3,7,11-trimethyl-1,3,6,10-dodecatetraene) that is either constitutively present or induced in a wide range of species.
The biosynthetic pathway for the sesquiterpenes branches off from the general terpenoid pathway, beginning with the allylic diphosphate ester farnesyl diphosphate (FDP, also shortened to FPP) (Bohlmann, et al., Proc. Natl. Acad. Sci. U.S.A. 95, 4126-4133 (1998), Cane and Bowser, Bioorg. Med. Chem. Lett. 9, 1127-1132 (1999), Davis and Croteau, Top. Curr. Chem. 209, 53-95 (2000)). Alpha-farnesene is synthesised from FDP in a reaction that proceeds through a carbocation intermediate (FIG. 2) and is catalysed by the sesquiterpene synthase alpha-farnesene synthase (Rupasinghe, et al., J. Am. Soc. Hortic. Sci. 123, 882-886 (1998)). The pathway for sesquiterpene biosynthesis, the acetate/mevalonate pathway, is localised to the cytoplasm; in contrast to the pathways for monoterpene and diterpene biosynthesis, which occur in the chloroplast (Croteau, et al., In Biochemistry and Molecular Biology of Plants, eds Buchanan, Gruissem and Jones, American Society of Plant Physiologists, 1250-1318 (2000); Lange, et al., Proc. Natl. Acad. Sci. U.S.A. 97, 13172-13177 (2000)).
All known plant terpene synthases, however, whether monoterpene, sesquiterpene or diterpene, appear to be closely related. Similarities include the positioning of intron sequences (Trapp and Croteau, Genetics 158, 811-832 (2001)) and the presence of conserved sequences, such as an aspartate-rich DDXX(D,E) motif (Lesburg, et al., Curr. Opin. Struct. Biol. 8, 695-703 (1998)). This motif is involved in the binding of metal ions, usually Mg2+, that are necessary for catalysis. (Lesburg, et al., Curr. Opin. Struct. Biol. 8, 695-703 (1998)).
Alpha-farnesene synthase has been partially purified from the skin of apple fruit (Malus domestica Delicious). However, poor recovery and instability of the partially purified enzyme restricted further purification (Rupasinghe, et al., J. Am. Soc. Hortic. 125, 111-119 (2000)).
Alpha-farnesene is an insect attractant. It is a sex pheromone in mice and insects. Oxygenated (including chemicals occurring on exposure to air) alpha-farnesene products (eg farnesol, farnesal) have characteristic aromas (flavour/fragrance use). Other uses for alpha-farnesene and its derivatives are as potent cancer prevention agents, and in plastic film synthesis.
There is also a link between both the levels of alpha-farnesene and its oxidation products and the development of superficial scald, a postharvest physiological disorder that appears as a dark coloration of the apple skin following cool storage (Watkins, et al., Acta Hort. 343, 155-160 (1993), Ju and Bramlage, J. Am. Soc. Hortic. Sci. 125, 498-504 (2000), Whitaker and Saftner, J. Agric. Food Chem. 48, 2040-2043 (2000), Rowan, et al., J. Agric. Food Chem. 49, 2780-2787 (2001)). To date the causal relationship between alpha-farnesene and scald is still unclear (Ju and Curry, J. Am. Soc. Hortic. Sci. 125, 626-629 (2000), Rupasinghe, et al., J. Am. Soc. Hortic. Sci. 125, 111-119 (2000)). Ethylene production and alpha-farnesene biosynthesis also appear to be closely associated (Watkins, et al., Acta Hort. 343, 155-160 (1993), Fan, et al., J. Agric. Food Chem. 47, 3063-3068 (1999)). Recently it has been shown that ethylene may regulate the biosynthesis of alpha-farnesene during fruit ripening by acting on the mevalonate pathway, specifically by inducing the conversion of hydroxymethylglutaryl CoA to mevalonic acid (Ju and Curry, J. Am. Soc. Hortic. Sci. 125, 105-110 (2000), Ju and Curry, Postharvest Biol. Technol. 19, 9-16 (2000), Ju and Curry, J. Am. Soc. Hortic. Sci. 126, 491-495 (2001)).
It is an object of the invention to provide methods for in vitro synthesis of alpha-farnesene and/or for genetically modifying plants to alter the levels of alpha-farnesene synthase activities in plants; and/or to offer the public a useful choice.